The field of this invention is the area of plant molecular biology, in particular the genetic engineering of plants to express chimeric genes determining resistance to heavy metals and/or thio-reactive metals, for example, a plant-expressible arsenate reductase coding sequence and/or a phytochelatin biosynthetic enzyme (and optionally, a mercuric ion reductase gene) and the use of such plants in the phytoremediation of environmental pollutants including arsenate, arsenite, cobalt, copper, zinc, mercury, platinum, palladium, antimonate and cadmium ions.
As a consequence of the industrial revolution there has been a significant increase in anthropogenic emission of heavy metals into the biosphere (Ayers, 1992). Mining, smelting, vehicle exhausts, and toxic run-off agricultural products are the source of most anthropogenic pollution of soils and sediments (Nriagu, 1980). These activities have led to solid contaminated particularly with heavy metals such as Cu, Zn, Pb, Ni, Cd, Hg and As. Cadmium is a toxic metal widely spread in the environment and in foods consumed by man (Sherlock, 1984). Major uses of cadmium include electroplating (35%), paint pigments (25%), plastic stabilizers (15%), and batteries (15%) (Nriagu, 1980). One serious soil-related pollution problem is the elevated level of cadmium in agricultural products such as phosphate fertilizers and cadmium containing sewage sludges (Nicholson and Jones, 1994); Ryan et al., 1982) and cadmium accumulation in plants grown on the cadmium-contaminated soil and sediments. Chronic exposure to such soils and consumption of contaminated food pose a serious threat to human and animal health (Sherlock, 1984). Cadmium exposure causes anemia, hypertension, hepatic, renal and cardiovascular disorders.
Physical remediation methods like soil removal and burial are impractical because the expense involved in large-scale removal is too great. Moreover, unlike organic waste, which can be mineralized, metals are immutable and can not be degraded into harmless constituents (Meagher, 2000). Phytoremediation, the use of green plants to remove toxic contaminants from soil and water, is an environmental friendly and cost effective solution for cleaning up metal contaminated sites and it has been suggested that plants might play a significant role in the phytoremediation of toxic heavy metals such as Hg, As, Cd, Cr, Zn, Ni, and Pb from contaminated soil and water (Raskin, 1996).
For a successful phytoremediation strategy, plants must be able to tolerate and hyperaccumulate the toxic metals aboveground (Goldsbrough, 1998). A number of plant species can hyperaccumulate metals in their aboveground tissues to levels far exceeding those present in soil or in the non-hyperaccumulating species growing nearby (Baker and Brooks, 1989). Hyperaccumulation is usually defined as levels of metal ions greater than 0.1%-1% of the dry weight of the plant (Baker, 2000). At these concentrations the recovery of metals from the plant tissues is potentially economical (Baker, 2000). Recovery of even low hyperaccumulated concentrations of most toxic metals such as As, Cd, and Hg could be economically viable as an alternative to the extreme expense of physical remediation methods. In the past few years, interest has been shown to exploit plants' natural properties to remediate toxic heavy metals soils through phytoextraction and phytomining (Brown et al., 1995b; McGrath et al., 1993); Robinson et al., 1997). Unfortunately, most of these hyperaccumulator species are small and slow growing, therefore, their potential for large scale remediation of polluted sites is limited (Ebbs et al., 1997).
Bacterial cells have developed more assertive solutions to exposure to environmental toxins (Meagher, 2000). Bacteria have evolved mechanisms to reduce, oxidize or modify metal ions to less toxic forms and eliminate these toxic metals from their cytoplasm by specific transporters (Tsai et al., 1997). One such system is the ars operon which provides resistance to arsenate and arsenite. Resistance to arsenate is acquired by first reducing it to arsenite by the action of the arsC protein in conjunction with glutathione.

Arsenite is then pumped from the cell by the arsA/arsB complex (Rosen, 1999). The manipulation of bacterial arsenic resistance gene may provide resistance to arsenate and other metal ions. Bacterial genes modified for plant expression have been successfully expressed in plants for phytoremediation purposes. Transgenic plants expressing modified bacterial merA and merB genes have been shown to efficiently extract of elemental and methylmercury from contaminated media (Bizily et al., 1999; Rugh et al., 1998; Rugh et al., 1996). Similarly, transgenic Indian mustard plants overexpressing E. coli thio-rich peptides showed enhanced accumulation and tolerance to Cd (Zhu et al., 1999a; Zhu et al. 1999b).
Genetically engineered high biomass transgenic plants expressing genes responsible for hyperaccumulation hold potential for making phytoremediation a viable commercial technology (Brown et al., 1995a; Rugh et al., 1998). Higher plants can extract pollutants from the soil or water through their root systems, store and concentrate the pollutants in their cells and/or convert toxic pollutants to less toxic forms. Roots are more directly in contact with heavy metals in the environment than shoots and root growth usually responds more rapidly to metal exposure than shoot growth. Plants may produce up to 100×106 miles of roots per acre and thus plants are in contact with a vast expanse of soil surface area (Dittmer, 1937). Plant roots can also reach reasonable depths into the soil surface area (Stone and Kalisz, 1991). Therefore, one phytoremediation approach for heavy metals is to use plant roots to extract, the vascular system to transport, and leaves as a sink to concentrate the heavy metals aboveground. Our initial aim was to study whether ArsC protein reduces other structurally related metal ions. Herein, we disclose that the expression of arsC gene modified for expression in plants enables bacterial cells and transgenic plants including, but not limited to, transgenic Arabidopsis plants to grow on otherwise toxic levels of Cd(II) by reducing it to Cd(0).
There is a long felt need in the art for the in situ remediation of toxic metal ions including but not limited to arsenate; mercury, arsenite, antimony, zinc, copper, cobalt, platinum, palladium, and cadmium ions and ion complexes thereof. The present invention enables phytoremediation and/or revegetation of contaminated environments, especially those contaminated with cadmium, via the plant-expressible arsenate reductase and/or phytochelatin biosynthetic sequences and/or mercury reductase coding sequences disclosed herein.